The proposed experiments are designed to identify which of three possible adenovirus type 9 (Ad9)E4 proteins is required for eliciting mammary tumors in rats and to characterize the identified oncogenic Ad9 E4 protein with respect to functional domains and possible physical interactions with cell proteins. E4 region recombinant adenoviruses between Ad9 and Ad26, a related non-tumorigenic virus, will be constructed for determination of their mammary gland oncogenicity in rats. These experiments will define a single Ad9 E4 region open reading frame (ORF) essential to mammary tumor production, and Ad9 E4 region mutant viruses will demonstrate that expression of the identified Ad9 E4 ORF protein is necessary for mammary gland oncogenesis. Further experiments will employ PCR mutagenesis to incorporate unique Ad9 amino acids into the Ad26 E4 protein, and vice versa. Such recombinant E4 protein ORFs, built into viruses, will be used to identify Ad9 E4 protein amino acids/domains necessary for mammary tumor production. Other experiments will attempt to identify an in vitro mammary cell culture system which mirrors the oncogenicity of Ad9 and non-oncogenicity of Ad26 in rats. Such a system will reduce the number of animals and facilitate E4 protien biochemical analyses in the proposed experiments. Finally, since DNA tumor virus oncoproteins frequently heterocomplex with cellular proteins, immunoprecipitation and association with purified Ad9 E4 fusion protein will be used to assess heterocomplex formation by the oncogenic Ad9 E4 protein. If heterocomplexes are found, a mammary tumor line expression library will be screened with a radioactively labelled Ad9 E4 protein, or alternatively heterocomplexes will be purified and microsequenced, in order to clone a cDNA for a specifically associated cellular protein. The E4 gene of Ad9 is the first known viral gene specifically involved with eliciting a mammary tumor, and the mammary tumors produced by Ad9 in rats represent the most common type of breast tumor encountered in women under 35 years of age. Thus, the study of this viral system has potential for identifying cellular proteins which, when perturbed, lead to a common breast cancer. Identifying such proteins will provide insights into the unique susceptibility of the mammary gland to cancer and will be important for designing better treatments and eventually preventing this significant disease.